Eleni Nastouli

Full-Genome Deep Sequencing and Phylogenetic Analysis of Novel Human Betacoronavirus

A novel betacoronavirus associated with lethal respiratory and renal complications was recently identified in patients from several countries in the Middle East. We report the deep genome sequencing of the virus directly from a patient’s sputum sample. Our high-throughput sequencing yielded a substantial depth of genome sequence assembly and showed the minority viral variants in the specimen. Detailed phylogenetic analysis of the virus genome (England/Qatar/2012) revealed its close relationship to European bat coronaviruses circulating among the bat species of the Vespertilionidae family. Molecular clock analysis showed that the 2 human infections of this betacoronavirus in June 2012 (EMC/2012) and September 2012 (England/Qatar/2012) share a common virus ancestor most likely considerably before early 2012, suggesting the human diversity is the result of multiple zoonotic events.

H1N1 pneumonitis treated with intravenous zanamivir

On July 8, 2009, a 22-year-old woman, neutropenic after chemotherapy for Hodgkin’s disease, was referred to ICU with 3 days’ (d) increasing dyspnoea, bilateral chest in fi ltrates, and laboratory-confi rmed pandemic H1N1 2009 infl uenza virus infection not responding to oseltamivir 75 mg twice daily and broad-spectrum antimicrobials (mero penem, teicoplanin, and caspofungin). No other organisms were detected from blood or respiratory tract. Deterioration necessitated invasive ventilation from ICU d 3 (fi gure). She remained in single organ failure requiring high inspired oxygen, protective lung ventilation (tidal volumes ≤6–8 mL/kg), and neutral fl uid balance. Hydro cortisone was given (d 3–6), then gradually reduced and discontinued (d 13). Neutropenia recovered by d 6, although lymphopenia remained (webappendix). High level H1N1 RNA was detected in bronchoalveolar lavage (BAL) on d 10, despite 6 d oseltamivir given nasogastrically; in view of high volume gastric aspirates, this was replaced by nebulised zanamivir (d 6–13). Treatment escalation on d 13–16 delivered neither clinical nor virological response (fi gure). On d 16, intravenous zanamivir 600 mg twice daily (provided by GlaxoSmithKline, Brentford, Middlesex) was started as unlicensed antiviral monotherapy; agreement for use was granted by the Hospital Formulary Committee and next of kin. Methylprednisolone was also started. Our patient’s condition improved within 48 h, with a decrease in BAL viral load on d 21. She was extubated on d 21 and discharged to the ward on d 24. Antiviral and steroid treatment were stopped on d 26 and d 28, respectively. Since ICU discharge she remains stable. Of four nasopharyngeal swabs taken post-ICU, the third, taken on d 10 post-ICU, showed H1N1 RNA Ct of 24, although a repeat sample taken the next day was negative. In view of her immunosuppressed state and ongoing lymphopenia, inhaled zanamivir was started as a precaution, although her clinical status remained unchanged. Deaths due to pandemic H1N1 are primarily related to severe respiratory failure. Our patient did not respond to exten sive antiviral treatment and 2 weeks’ mechanical ventilation. RT-PCR detects viral RNA rather than infectious virus, but is used to semi-quantitatively assess replication. The small diff erence in Ct between d 10 and 16 implied continued high-level replication. Eff ective treat ment depends on adequate enteral absorption (oseltamivir) and an uninhibited access to the infected respira tory tissue (zanamivir). In view of high volume gastric aspirates, we used nebulised zanamivir. Since her infl amed, atelectatic lungs were probably im peding adequate drug absorption, and clinical improve ment was not forth coming, we used intravenous (un licensed) zanamivir. High dosing achieves eff ective respira tory epithelial concentrations and is well-tolerated. Our pa tient recovered with no side-eff ects. Despite in herent sampling inconsistencies, the change in BAL Ct from 23 to 30 after 5 d treatment indicates an approximate 128-fold fall in viral load. Persisting high-level H1N1 replication may drive ongoing lung infl ammation and fi brosis (im plied by our patient’s poor lung compliance). We reasoned that synergism could exist between intra venous zanamivir and high-dose corticosteroids, al though this approach may be considered controversial and is not recom mended in treatment guide lines. However, con trolled trials are lacking and a rationale does exist for the use of corticosteroids in ARDS. Although this is a single case report and direct cause and eff ect cannot be confi rmed, the im prove ment in clinical status following intravenous zanamivir encourages prompt further investigation, both alone and in com bination with high-dose methyl pred nisolone.

The Third Signal Cytokine IL-12 Rescues the Anti-Viral Function of Exhausted HBV-Specific CD8 T Cells

Optimal immune activation of naïve CD8 T cells requires signal 1 mediated by the T cell receptor, signal 2 mediated by co-stimulation and signal 3 provided by pro-inflammatory cytokines. However, the potential for signal 3 cytokines to rescue anti-viral responses in functionally exhausted T cells has not been defined. We investigated the effect of using third signal cytokines IL-12 or IFN-α to rescue the exhausted CD8 T cell response characteristic of patients persistently infected with hepatitis B virus (HBV). We found that IL-12, but not IFN-α, potently augmented the capacity of HBV-specific CD8 T cells to produce effector cytokines upon stimulation by cognate antigen. Functional recovery mediated by IL-12 was accompanied by down-modulation of the hallmark inhibitory receptor PD-1 and an increase in the transcription factor T-bet. PD-1 down-regulation was observed in HBV but not CMV-specific T cells, in line with our finding that the highly functional CMV response was not further enhanced by IL-12. IL-12 enhanced a number of characteristics of HBV-specific T cells important for viral control: cytotoxicity, polyfunctionality and multispecificity. Furthermore, IL-12 significantly decreased the pro-apoptotic molecule Bim, which is capable of mediating premature attrition of HBV-specific CD8 T cells. Combining IL-12 with blockade of the PD-1 pathway further increased CD8 functionality in the majority of patients. These data provide new insights into the distinct signalling requirements of exhausted T cells and the potential to recover responses optimised to control persistent viral infections.

Comparative Study of Sensitivity, Linearity, and Resistance to Inhibition of Digital and Nondigital Polymerase Chain Reaction and Loop Mediated Isothermal Amplification Assays for Quantification of Human Cytomegalovirus

Performing nucleic acid amplification techniques (NAATs) in digital format using limiting dilution provides potential advantages that have recently been demonstrated with digital polymerase chain reaction (dPCR). Key benefits that have been claimed are the ability to quantify nucleic acids without the need of an external calibrator and a greater resistance to inhibitors than real-time quantitative PCR (qPCR). In this study, we evaluated the performance of four NAATs, qPCR, dPCR, real-time quantitative loop mediated isothermal amplification (qLAMP), and digital LAMP (dLAMP), for the detection and quantification of human cytomegalovirus (hCMV). We used various DNA templates and inhibitors to compare the performance of these methods using a conventional real-time thermocycler platform (Bio-Rad CFX96) and a chip based digital platform (Fluidigm Biomark 12.765 Digital Array). dPCR performed well and demonstrated greater resistance to inhibitors than the other methods although this resistance did not apply equally to all inhibitors tested. dLAMP was found to be less sensitive than dPCR, but its quantitative performance was better than qLAMP, the latter being unable to quantify below 1000 copies. dLAMP was also more resistant to inhibitors than qLAMP. Unlike qPCR, both digital methods were able to quantify viral genomes without requiring a calibrator; however, neither can currently compete with the large reaction volumes, and thus the greater absolute sensitivity, of qPCR. With the introduction of digital instrumentation that will enable larger reaction volumes, digital amplification methods such as those evaluated in this study could potentially offer a robust alternative to qPCR for nucleic acid quantification.

IVA: accurate de novo assembly of RNA virus genomes

Motivation: An accurate genome assembly from short read sequencing data is critical for downstream analysis, for example allowing investigation of variants within a sequenced population. However, assembling sequencing data from virus samples, especially RNA viruses, into a genome sequence is challenging due to the combination of viral population diversity and extremely uneven read depth caused by amplification bias in the inevitable reverse transcription and polymerase chain reaction amplification process of current methods. Results: We developed a new de novo assembler called IVA (Iterative Virus Assembler) designed specifically for read pairs sequenced at highly variable depth from RNA virus samples. We tested IVA on datasets from 140 sequenced samples from human immunodeficiency virus-1 or influenza-virus-infected people and demonstrated that IVA outperforms all other virus de novo assemblers. Availability and implementation: The software runs under Linux, has the GPLv3 licence and is freely available from http://sanger-pathogens.github.io/iva Contact: iva@sanger.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online.

Minor groove binder modification of widely used TaqMan probe for hepatitis E virus reduces risk of false negative real-time PCR results

Hepatitis E virus (HEV) is a major cause of acute viral hepatitis in many parts of the developing world. It is responsible for both sporadic infections and large scale epidemics and may be associated with significant mortality during pregnancy. Over the past two decades many serological and nucleic acid based diagnostic tests for HEV have been developed, including several reverse transcription real-time polymerase chain reaction assays (RT-qPCR). One of the most widely used of these RT-qPCRs is that developed by Jothikumar and colleagues (Journal of Virological Methods 2006, 131, 65-71). Whilst reviewing this assay we calculated the predicted melting temperature of its TaqMan probe and consequently synthesised a minor groove binder (MGB) version in order to increase its hybridisation stability. In this report the performance of the original unmodified probe is compared with that of the MGB-modified version. We demonstrate that the MGB-modified probe detected HEV RNA in plasma samples from six patients with serologically confirmed hepatitis E in whom the unmodified probe had failed to detect HEV RNA. Sequence analysis of the ORF3 segment targeted by the RT-qPCR was possible in 4 of the 6 patients and revealed an identical C→T single nucleotide mutation in the probe binding region in each case.

Quantitation of hepatitis delta virus using a single-step internally controlled real-time RT-qPCR and a full-length genomic RNA calibration standard.

Quantitation of circulating hepatitis delta virus (HDV) RNA is important for assessing the response to antiviral therapy and for understanding the complex dynamic interactions between hepatitis B virus (HBV) and HDV replication. Although several PCR assays for HDV RNA have been described none of them incorporate an internal control or use a full-length RNA calibration standard for absolute quantitation. This study describes the development and evaluation of a novel single-step real-time RT-qPCR assay for HDV RNA quantitation which incorporates a Brome Mosaic virus internal control to prevent false negatives and under-reporting due to inhibitors or due to inefficient RNA purification, reverse transcription or PCR amplification. The assay has a dynamic range of ≥7log(10) and is designed to detect all HDV genotypes. The 95% detection limit is ∼3800 HDV RNA copies/ml, 700 copies/ml being detectable in 20% of repeats. Both intra-assay and inter-assay variability are low (CV 8% and 17%, respectively). Plasma HDV RNA was detected in 75% of 59 HDV antibody-positive samples with titres ranging from 8.4×10(4) to 4.4×10(8) copies/ml. The assay described provides a reliable and sensitive quantitative system for therapeutic monitoring and for studying the dynamic interplay between hepatitis B virus replication and HDV viral load.

Distinct Metabolic Requirements of Exhausted and Functional Virus-Specific CD8 T Cells in the Same Host

Summary T cells undergo profound metabolic changes to meet the increased energy demands of maintaining an antiviral response. We postulated that differences in metabolic reprogramming would shape the efficacy of CD8 T cells mounted against persistent viral infections. We found that the poorly functional PD-1hi T cell response against hepatitis B virus (HBV) had upregulated the glucose transporter, Glut1, an effect recapitulated by oxygen deprivation to mimic the intrahepatic environment. Glut1hi HBV-specific T cells were dependent on glucose supplies, unlike the more functional cytomegalovirus (CMV)-specific T cells that could utilize oxidative phosphorylation in the absence of glucose. The inability of HBV-specific T cells to switch to oxidative phosphorylation was accompanied by increased mitochondrial size and lower mitochondrial potential, indicative of mitochondrial dysfunction. Interleukin (IL)-12, which recovers HBV-specific T cell effector function, increased their mitochondrial potential and reduced their dependence on glycolysis. Our findings suggest that mitochondrial defects limit the metabolic plasticity of exhausted HBV-specific T cells.

Persistent Zika Virus Detection in Semen in a Traveler Returning to the United Kingdom from Brazil, 2016.

Zika virus is normally transmitted by mosquitos, but cases of sexual transmission have been reported. We describe a patient with symptomatic Zika virus infection in whom the virus was detected in semen for 92 days. Our findings support recommendations for 6 months of barrier contraceptive use after symptomatic Zika virus infection.

Detection of rare drug resistance mutations by digital PCR in a human influenza A virus model system and clinical samples

ABSTRACT Digital PCR (dPCR) is being increasingly used for the quantification of sequence variations, including single nucleotide polymorphisms (SNPs), due to its high accuracy and precision in comparison with techniques such as quantitative PCR (qPCR) and melt curve analysis. To develop and evaluate dPCR for SNP detection using DNA, RNA, and clinical samples, an influenza virus model of resistance to oseltamivir (Tamiflu) was used. First, this study was able to recognize and reduce off-target amplification in dPCR quantification, thereby enabling technical sensitivities down to 0.1% SNP abundance at a range of template concentrations, a 50-fold improvement on the qPCR assay used routinely in the clinic. Second, a method was developed for determining the false-positive rate (background) signal. Finally, comparison of dPCR with qPCR results on clinical samples demonstrated the potential impact dPCR could have on clinical research and patient management by earlier (trace) detection of rare drug-resistant sequence variants. Ultimately this could reduce the quantity of ineffective drugs taken and facilitate early switching to alternative medication when available. In the short term such methods could advance our understanding of microbial dynamics and therapeutic responses in a range of infectious diseases such as HIV, viral hepatitis, and tuberculosis. Furthermore, the findings presented here are directly relevant to other diagnostic areas, such as the detection of rare SNPs in malignancy, monitoring of graft rejection, and fetal screening.